Methods and compositions for the treatment and prevention of Staphylococcus a ureus infections

ABSTRACT

The invention features methods and compositions for treatment or prevention of infection by, or disease caused by infection with, Staphylococcus spp., particularly  S. aureus.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/054,331, filed Apr. 2, 1998, which application claims thebenefit of U.S. provisional application serial No. 60/068,094, filedDec. 19, 1997, now abandoned, each of which applications is incorporatedby reference in its entirety herein for all purposes.

TECHNICAL FIELD

[0002] The present invention relates to methods and compositions fortreatment or prevention of bacterial infection, diseases or symptomscaused by bacterial infection, and particularly those associated withinfection by Staphylococcus spp.

BACKGROUND OF THE INVENTION

[0003]Staphylococcus aureus causes disease chiefly through theproduction of virulence factors such as hemolysins, enterotoxins andtoxic shock syndrome toxin. The synthesis of virulence factors in S.aureus is controlled by a regulatory RNA molecule, RNAIII (Novick,etal., EMBO J. 12, 3967 (1993), Balaban et al., FEMS Microbiol Letts.133, 155 (1995), Moerfeldt et al., EMBO J. 14, 4569 (1995)), encoded bythe agr locus. The rnaiii gene of the agr locus is transcribed inculture only from the midexponential phase of growth, and is autoinducedby the protein RNAIII activating protein (RAP) (Balaban et al., Proc.Natl. Acad. Sci. USA. 92,1619 (1995)). RAP is continuously secreted bythe bacteria and only activates RNAIII at a concentration threshold(ibid).

[0004] The growth-phase associated regulation of S. aureus virulencefactor synthesis is controlled by a quorom sensing mechanism. Thecontrol of virulence factor production is a complex process, whichapparently involves multiple global regulatory loci. One of theseregulatory loci, the agr locus, contains two divergent transcriptionunits, RNAII and RNAIII, both of which are active only from themidexponential phase of growth and are autocatalytic (Novick et al. Mol.Gen. Genet. 248:446058 (1995)). RNAIII, an RNA regulatory moleculeencoded by the agr locus, upregulates genes encoding for toxicexomolecules while down regulating genes encoding for surface molecules,resulting in vivo in dissemination and disease (Novick et al. EMBO J.12:3967-75 (1993); Balaban et al. Proc. Natl. Acad. Sci. USA 92:1619-23(1995); Moerfeldt et al. EMBO J. 14:4569-77 (1995)). The RNAII locusregulates the expression of RNAIII. The RNAII locus comprises four openreading frames (ORFs), agrA, agrB, agrC, and agrD. The agrA and agrCgenes encode for a classical two-component signal transduction pathway,with agrC encoding a signal receptor and agrA the response regulator.

[0005] The autoinducers of RNAIII that have been described to dateinclude the agr-independent RNAIII activating protein (RAP) (Balaban etal (1995) supra), and the agrD-derived octapeptide pheromone (Ji et al.Proc. Natl. Acad. Sci. 92:12055-9 (1995)). The agrD-derived octapeptidehas also been shown to be part of a “bacterial interference” system thatprovides a mechanism for different S. aureus strains to compete witheach other at an infection site (Ji et al. Science 276:2027-30 (1997)).In this bacterial interference system, the octapeptide activates RNAIIItranscription of the strain by which it is produced, while also actingas an inhibitor of RNAIII transcription of other strains ofStaphyloccocus.

[0006] In addition to agr, the sar locus also plays a role in regulationof S. aureus virulence factor production. The sar locus comprises a sarAORF preceded by a triple promoter region interspersed with two putativesmaller ORFs (ORF3 and ORF4). The triple promoter system yields threeoverlapping sar transcripts (sarA, sarC and sarB) (Bayer et al. J.Bacteriol. 178:4563-70 (1996)).

[0007] In vivo S. aureus first produce proteins that facilitatebacterial binding to host cells as well as the secreted autoinducermolecules. As the bacterial colony increases in density, the autoinducermolecules accumulate. Upon reaching a threshold concentration, theautoinducers activate RNAIII transcription, which in turn results invirulence factor production. The virulence factors damage and eventuallydestroy surrounding host cells, which serve as nutritive sources for theS. aureus bacteria and promoting further growth of the colony. Thus,inhibition of RNAIII by suppression of the autoinducers or theirreceptors is of particular interest in treatment or prevention of S.aureus-mediated disease. Several mechanisms for RNAIII inhibition havebeen identified, including inhibition of RNAIII by anti-RAP antibodiesand by a peptide termed the RNAIII inhibiting peptide (RIP), whichcompetes with RAP (Balaban et al. (1995) supra).

[0008]S. aureus causes diseases ranging from minor skin infections tolife-threatening deep infections such as pneumonia, endocarditis,meningitis, postoperative wound infections, septicemia, and toxic shocksyndrome (Silverstein et al., in Microbiology, Davis et al., eds.(Lippincott, Philadelphia, 1990), pp. 485-506). Hospitalized patientsare at particular risk, with over 500,000 nosocomial infections per year(Panlilio, et al., Inf. Contr.and Hasp. Epidem. 13, 582 (1992)). Theemergence of drug resistance has made many of the availableantimicrobial agents ineffective. Therefore, alternative methods for theprevention and treatment of bacterial infections in general and S.aureus infections in particular are eagerly sought. The instantinvention addresses this need and others.

SUMMARY OF THE INVENTION

[0009] The invention features methods and compositions for treatment orprevention of infection by, or disease caused by infection with,Staphylococcus spp., particularly S. aureus.

[0010] One aspect of the invention is a composition comprising apolypeptide comprising an amino acid sequence comprising the generalformula Y(K or S) PXTNF (SEQ ID NOS: 1 and2), where X is C, W, or I.Pharmaceutical compositions are also provided in some embodiments. Afurther aspect of the invention is a composition of claim 1, wherein thepolypeptide comprises an amino acid sequence comprising the generalformula IKKY(K or S)PXTNF (SEQ ID NOS:3 and 4), where X is C, W, or I.

[0011] A further aspect of the invention is a method for treating a hostfor a staphylococcal infection, wherein the composition of claim 1 isadministered to the host. In some embodiments the host is a humanpatient. In further embodiments the host is an animal, such as but notlimited to an experimental animal.

[0012] A further aspect of the invention is a method for treating a hostfor a staphylococcal infection, wherein an antagonist of the RAPreceptor is administered to the host. In some embodiments the host is ahuman patient. In further embodiments the host is an animal, such as butnot limited to an experimental animal. In some embodiments theantagonist is a polypeptide, a peptidomimetic, or an antibody.

[0013] A further aspect of the invention is a nucleic acid moleculeencoding a polypeptide of the invention. The nucleic acid molecule canbe RNA or DNA or an antisense nucleic acid molecule. In an embodiment,the nucleic acid molecule comprises the nucleotide sequence TAT TCG CCGTGG ACC AAT TTT (SEQ ID NO:5).

[0014] In another aspect, the invention features an isolated RAPpolypeptide, as well as nucleic acid encoding such RAP polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1A is a graph depicting an assay for RNAIII activation asdescribed in Balaban et al. (supra). Post-exponential supernatants ofwild-type S. aureus (denoted total) were filtered through a 3-kD cutoffmembrane and the flow-through containing agrD-encoded octapeptide(Guangyong, et al, Proc. Natl. Acad. Sci. U.S.A. 92, 12055 (1995))collected (denoted <3-kD). Retained material (containing RAP) wasfiltered through a 10-kD cutoff membrane. Material greater than 10-kD(denoted >10-kD) was applied to an HPLC gel filtration column andpurified RAP was collected (RAP). Increasing amounts of each of thesecompositions were added to early exponential wild type S. aureus andtested for their ability to activate RNAIII.

[0016]FIG. 1B is a graph depicting the ability of 1 ml fractionscollected from postexponential supernatants of wild type S. aureus andof arg-null S. aureus fractionated on a gel filtration to activateRNAIII.

[0017]FIG. 1C is a graph depicting the absorbance at A₂₈₀ of fractionsfrom FIG. 1B containing peak RNAIII-inducing activity.

[0018]FIG. 1D is a photograph of gel-filtration-purified RAP from wildtype (lane 1) and from agr-null strain (lane 2) separated on SDS PAGEand silver stained. Approximate molecular weight markers are indicated.

[0019]FIG. 2A is a photograph of an immunoblot of sera of vaccinated andcontrol animals. Postexponential supernatant of wild type S. aureus(Lanes 1,2) or purified RAP (Lane 3) was separated on SDS 12% PAGE,western blotted, and membranes were incubated in the presence of: Lane1: pre-immune (lane 1a) or post-immune (lane Ib) sera collected from acontrol CFA-injected animal (diluted 1:20). Lane 2: pre-immune sera(lane 2a, diluted 1:20) or post immune sera (lane 2b diluted 1:1000 andlane 3 diluted 1:20). Approximate molecular weight markers are indicatedin kilodaltons.

[0020]FIG. 2B is a graph depicting the titer of anti-RAP antibodies vslesion size of vaccinated animals.

[0021]FIG. 3 is a graph depicting the inhibition of RNAIII by purifiedand synthetic RIP.

[0022]FIGS. 4A and 4B are graphs depicting the inhibition of RNAIII bysynthetic RIP peptides.

[0023]FIG. 4C is a graph depicting competition between RAP and purifiedor synthetic RIP.

[0024]FIG. 5 is a schematic illustrating the DNA and amino acidsequences of RAP.

[0025]FIGS. 6A and 6B are photograph of gels showing purification ofrecombinant RAP (rRAP) eluted from a nickel column by 1M (lane 1), 2M(lane 2) and 3M (lane 3) imidazole applied to SDS 12.5% PAGE. The gelwas Western blotted, membrane stained in ponceau to visualize proteins(FIG. 6A), blocked in milk, and incubated with anti-histidine antibodies(FIG. 6B). Bound antibodies were detected by peroxidase-conjugatedanti-mouse antibodies, and visualized by ECL (Amersham). Molecular massis indicated in kDa.

[0026]FIG. 7 is a graph showing the percent mortality of Balb/c naivemice (control) or mice vaccinated with rL2, and challenged with 2×10⁹ S.aureus.

[0027]FIG. 8 is a graph showing the development of lesions in rL2vaccinated animals that survived a challenge of 2×10⁹ S. aureus.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Before the present proteins, formulations and methods aredescribed, it is to be understood that this invention is not limited tothe particular compounds, characteristics and steps described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

[0029] All publications and patents mentioned herein are incorporatedherein by reference to disclose and describe the specific methods and/ormaterials in connection with which the publications and patents arecited. The publications and patents discussed herein are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that thepresent invention is not entitled to antedate such publication or patentby virtue of prior invention. Further, the dates of publication orissuance provided may be different from the actual dates which may needto be independently confirmed.

[0030] Generally, the nomenclature used hereafter, and the laboratoryprocedures in cell culture and protein biochemistry are those well knownand commonly employed in the art. Generally, enzymatic reactions andcolumn chromatography are performed according the manufacturer'sspecifications. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are described. For the purposes of thepresent invention, the foregoing terms are defined below. The terms“pharmaceutically acceptable” or “therapeutically acceptable” refer to asubstance which does not interfere with the effectiveness or thebiological activity of the active ingredients and which is not toxic tothe host or the patient.

[0031] The terms “encoding” or “encodes” refer generally to the sequenceinformation being present in a translatable form, usually operablylinked to a promoter. A sequence is operably linked to a promoter whenthe functional promoter enhances transcription or expression of thatsequence. An anti-sense strand is considered to also encode thesequence, since the same informational content is present in a readilyaccessible form, especially when linked to a sequence which promotesexpression of the sense strand. The information is convertible using thestandard, or a modified, genetic code. See, e.g. Watson et a/., (1987)The Molecular Biology of the Gene. (4th Edition), Vols. 1 & 2, Benjamin,Menlo Park, Calif.

[0032] As used to refer to nucleic acid sequences, the term “homologous”indicates that two or more nucleotide sequences share a majority oftheir sequence. Generally, this will be at least about 70% of theirsequence and preferably at least 95% of their sequence.

[0033] Another indication that sequences are substantially identical isif they hybridize to the same nucleotide sequence under stringentconditions (see, e.g., Sambrook etal., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1985).Stringent conditions are sequence-dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (T{circumflex over( )} for the specific sequence at a defined ionic strength and pH. TheT_(m) is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.2 molar at pH 7 and the temperature isat least about 60° C.

[0034] As used to refer to proteins, polypeptides, or peptides, whichterms are used interchangeably here, the term “homologous” is meant toindicate two proteins or polypeptides'share a majority of their aminoacid sequences. Generally, this will be at least 90% and usually morethan about 95%. Homology for polypeptides or proteins is typicallymeasured using sequence analysis software, see e.g. Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison Wis. 53705.Protein analysis software matches similar sequences using measure ofhomology assigned to various substitutions, deletions, and othermodifications. Conservative substitutions typically includesubstitutions within the following groups glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; senne, threonine; lysine, arginine; and phenylalanine,tyrosine.

[0035] As used herein the term “isolated” is meant to describe acompound of interest (e.g., either a polynucleotide or a polypeptide)that is in an environment different from that in which the compoundnaturally occurs. “Isolated” is meant to include compounds that arewithin samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

[0036] The term “isolated” as applied to, for example, nucleic acids,means a nucleic acid substantially separated from other macromolecules,cellular components, or DNA sequences which naturally accompany a nativenucleic acid, e.g. ribosomes, polymerases, other nucleic acid sequences,and the like. The term includes a nucleic acid or polypeptide that hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogues,and analogues biologically synthesized by heterologous systems. Asubstantially pure or biologically pure nucleic acid includes isolatedforms of the nucleic acid.

[0037] The phrase “biologically pure” or “substantially pure” refers tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state, e.g., at least 60%free, preferably 75% free, and most preferably 90% free from othercomponents with which it is naturally associated.

[0038] The term “recombinant” refers to a nucleic acid sequence which isnot naturally occurring, or is made by the artificial combination of twootherwise separated segments of sequence, i.e. by chemical synthesis,genetic engineering, and the like.

[0039] The term “treatment” or “treating” means any therapeuticintervention in a mammal, preferably a human or bovine, including:

[0040] (i) prevention, that is, causing the clinical symptoms not todevelop, e.g., preventing infection from occurring and/or developing toa harmful state;

[0041] (ii) inhibition, that is, arresting the development of clinicalsymptoms, e.g., stopping an ongoing infection so that the infection iseliminated completely or to the degree that it is no longer harmful;and/or

[0042] (iii) relief, that is, causing the regression of clinicalsymptoms, e.g., causing a relief of fever and/or inflammation caused byan infection.

[0043] Treatment is generally applied to any mammal susceptible to ofhaving an S. aureus infection (e.g., mammals, birds, etc.), generally amammal, usually a human or bovine where the treatment can be applied forprevention of bacterial infection of for amelioration of activebacterial infection, where the bacteria is a Staphylococcus bacteria,specifically Staphylococcus aureus.

[0044] The terms “effective amount” and/or “therapeutic amount” means adosage sufficient to provide treatment for the disease state beingtreated. This will vary depending on the patient, the disease and thetreatment being effected. In the case of a bacterial infection, an“effective amount” is that amount necessary to substantially improve thelikelihood of treating the infection, in particular that amount whichimproves the likelihood of successfully preventing infection oreliminating infection when it has occurred.

[0045] The term “protein” as used herein is intended to encompass anyamino acid sequence and include modified sequences (e.g., glycosylated,PEGylated, containing conservative amino acid substitutions, etc.). Theterm includes naturally occurring (e.g., non-recombinant) proteins.polypeptides, peptides, (particularly those isolated from aStaphylococcus bacteria, more particularly from Staphylococcus aureus),and oligopeptides, as well as those which are recombinantly orsynthetically synthesized according to methods well known in the art. Asused in connection with the present invention the term “protein” isspecifically intended to cover naturally occurring proteins which occurin Staphylococcus spp.s and useful in treating infection or ingenerating antibodies useful in treating infection. Where “polypeptide”or “protein” are recited herein to refer to an amino acid sequence of anaturally-occurring protein molecule, “polypeptide,” “protein,” and liketerms are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.In addition, the polypeptides and proteins of the invention, orfragments thereof, can be generated in synthetic form having D-aminoacids rather than the naturally occurring L-amino acids.

[0046] “Polynucleotide” as used herein refers to an oligonucleotide,nucleotide, and fragments or portions thereof, as well as to peptidenucleic acids (PNA), fragments, portions or antisense molecules thereof,and to DNA or RNA of genomic or synthetic origin which can be single- ordouble-stranded, and represent the sense or antisense strand. Where“polynucleotide” is used to refer to a specific polynucleotide sequence(e.g. a RAP protein-encoding polynucleotide), “polynucleotide” is meantto encompass polynucleotides that encode a protein that is functionallyequivalent to the recited protein, e.g., polynucleotides that aredegenerate variants (i.e., variants in nucleic acid sequence that encodethe same amino acid sequence and exist due to the degeneracy of thegenetic code), or polynucleotides that encode biologically activevariants or fragments of the recited protein.

[0047] By “antisense polynucleotide” is meant a polynucleotide having anucleotide sequence complementary to a given polynucleotide sequenceincluding polynucleotide sequences associated with the transcription ortranslation of the given polynucleotide sequence (e.g, a promoter)and/or to a coding sequence of the given polynucleotide sequence, wherethe antisense polynucleotide is capable of hybridizing to apolynucleotide sequence. Of particular interest are antisensepolynucleotides capable of inhibiting transcription and/or translation,either in vitro or in vivo.

[0048] “Peptide nucleic acid” as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen et al1993 Anticancer Drug Des 8:53-63).

[0049] The term “antibody” is meant to refer to an immunoglobulinprotein which is capable of binding an antigen. Antibody as used hereinis meant to include the entire antibody as well as any antibodyfragments (e.g., F(ab)′, Fab, Fv) capable of binding the epitope,antigen or antigenic fragment of interest. Preferred antibodies forassays and vaccines of the invention are immunoreactive orimmunospecific for and therefore specifically and selectively bind to aprotein of interest, e.g., an anti-RAP antibody. The term “antibody”encompasses all types of antibodies, e.g., polyclonal, monoclonal,humanized, chimeric, and those produced by the phage displaymethodology. Particularly preferred antibodies of the invention areantibodies which have a relatively high degree of affinity for RAP. Anantibody of the invention is preferably immunoreactive with andimmunospecific for a specific species, e.g., RAP obtained fromStaphylococcus aureus.

[0050] “Antigenic fragment” of a protein is meant a portion of such aprotein which is capable of binding an antibody.

[0051] By “binds specifically” is meant high avidity and/or highaffinity binding of an antibody to a specific polypeptide, e.g., epitopeof a protein, e.g., RAP protein. Antibody binding to its epitope on thisspecific polypeptide is preferably stronger than binding of the sameantibody to any other epitope, particularly those which may be presentin molecules in association with, or in the same sample, as the specificpolypeptide of interest, e.g., binds more strongly to epitope fragmentsof a protein such as RAP so that by adjusting binding conditions theantibody binds almost exclusively to an epitope site or fragments of adesired protein.

[0052] By “detectably labeled antibody” is meant an antibody (orantibody fragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels known in the art include radioisotopes, fluorophores,paramagnetic labels, enzymes (e.g., horseradish peroxidase), or othermoieties or compounds which either emit a detectable signal (e.g.,radioactivity, fluorescence, color) or emit a detectable signal afterexposure of the label to its substrate. Various detectablelabel/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine,avidin/streptavidin, luciferase/luciferin), methods for labelingantibodies, and methods for using labeled antibodies are well known inthe art (see, for example, Harlow and Lane, eds. (Antibodies: ALaboratory Manual (1988) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.)).

[0053] The instant invention provides polypeptides for the preventionand treatment of S. aureus infections. These polypeptides comprise thegeneral formula Y(K or S)PXTNF (SEQ ID NOS: 1 and 2), where X is C, W,or I, preferably W. In a further embodiment, the polypeptides may havethe general formula IKKY(K or S) PXTNF (SEQ ID NOS:3 and 4), where X isC, W, or I, preferably W. The polypeptides are preferably at least 10amino acids in length, more preferably at least seven amino acids inlength.

[0054] Nucleic acids encoding the polypeptides of the invention are alsoincluded in the scope of the invention. Such nucleic acids may be DNA,RNA, or antisense nucleic acids. In an embodiment an isolated DNAmolecule of the invention comprises the sequence TAT TCG CCG TGG ACC AATTTT (SEQ IDNO:5). The nucleic acid molecules of the invention may beprovided as synthetic or purified, isolated molecules, including but notlimited to “naked DNA”; in vectors such as but not limited to plasmidsor viruses, including expression vectors, or complexed to othercompounds for administration. Such techniques are well known in the artThe polypeptides of the invention are preferably synthesized de novo byany technique commonly known in the art or may be encode;; by nucleicacid, such as RNA or DNA, delivered to the host. Purification fromcultures of S. aureus bacteria is discussed in the Experimental sectionbelow.

[0055] The polypeptides of the invention are typically administered tohosts having or at risk of having a staphylococcal infection such as anS. aureus infection. The hosts are typically human patients. Animals mayalso be treated with the compositions of the invention, including butnot limited to animals of commercial or veterinary importance such ascows, sheep, and pigs, and experimental animals such as rats, mice, orguinea pigs.

[0056] Typically, the compositions of the invention are administered ona daily basis for at least a period of 1-5 days. As used herein,“therapeutic dose” is a dose which prevents, alleviates, abates, orotherwise reduces the severity of symptoms in a patient The compositionsof the invention may be used prophylactically to prevent staphylococcalinfections or may be therapeutically used after the onset of symptoms.In some embodiments, induction of the formation of antibodies to theadministered compound is desirable. In such instances, standardimmunization protocols used in the art are preferred. The compositionsadministered for immunization may optionally include adjuvants.

[0057] In some embodiments of the invention, antagonists of the RAPreceptor are provided. Without being limited to any one theory, RIP mayfunction by competing with RAP for binding to the RAP receptor, thusacting as an antagonist of the RAP receptor. Such antagonists includebut are not limited to antibodies which specifically bind to RAP;antibodies which specifically bind to a RAP ligand; ligands for RAP orRIP; antisense nucleic acids; and peptide, non-peptide, andpeptidomimetic analogs of RAP, RIP, and their ligands.

[0058] Antibodies can be synthetic, monoclonal, or polyclonal and can bemade by techniques well known in the art. For therapeutic applications,“human” monoclonal antibodies having human constant and variable regionsare often preferred so as to minimize the immune response of a patientagainst the antibody. Such antibodies can be generated by immunizingtransgenic animals which contain human immunoglobulin genes. SeeJakobovits et al. Ann NY Acad Sci 764:525-535 (1995). In connection withsynthetic and semi-synthetic antibodies, such terms are intended tocover but are not limited to antibody fragments, isotype switchedantibodies, humanized antibodies (e.g., mouse-human, human-mouse, andthe like), hybrids, antibodies having plural specificities, fullysynthetic antibody-like molecules, and the like.

[0059] As discussed below, antibodies can be screened for the ability toblock the binding of a ligand to RAP or RIP and/or for other properties,such as the ability to protect in vivo against S. aureus infection.

[0060] In some embodiments of the invention, antisense nucleic acidmolecules are used as antagonists of RAP. Antisense nucleic acidmolecules are complementary oligonucleotide strands of nucleic acidsdesigned to bind to a specific sequence of nucleotides to inhibitproduction of a targeted protein. These agents may be used alone or incombination with other antagonists.

[0061] The antisense antagonist may be provided as an antisenseoligonucleotide such as RNA (see, for example, Murayama et al. AntisenseNucleic Acid Drug Dev. 7:109-114 (1997)). Antisense sequences may alsobe provided in a viral vector, such as, for example, in hepatitis Bvirus (see, for example, Ji et al., J. Viral Hepat. 4:167-173 (1997));in adeno-associated virus (see, for example, xiao et al. Brain Res.756:76-83 (1997)); or in other systems including but not limited to anHVJ(Sendai virus)-liposome gene delivery system (see, for example,Kaneda et al Ann. N.Y. Acad. Sci. 811:299-308 (1997)); a “peptidevector” (see, for example, Vidal et al. CR Acad. Sci III 32):279-287(1997)); as a gene in an episomal or plasmid vector (see, for example,Cooper et al. Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yewetal. Hum Gene Ther. 8:575-584 (1997)); as a gene in a peptide-DNAaggregate (see, for example, Niidome et al., J. Biol. Chem.272:15307-15312 (1997)); as “naked DNA” (see, for example, U.S. Pat. No.5,580,859 and U.S. Pat. No. 5,589,466); and inlipidic vector systems(see, for example, Lee et al. Crit Rev Ther Drug Carrier Syst.14:173-206 (1997)).

[0062] Candidate antagonists of the RAP receptor can be screened forfunction by a variety of techniques known in the art and/or disclosedwithin the instant application, such as protection against S. aureusinfection in a mouse model. A multitude of appropriate formulations ofthe antagonists of the invention can be found in the formulary known toall pharmaceutical chemists: Remington's Pharmaceutical Sciences, (15thEdition, Mack Publishing Company, Easton, Pa. (1975)), particularlyChapter 87, by Blaug, Seymour, therein. These formulations include forexample, powders, pastes, ointments, jelly, waxes, oils, lipids,anhydrous absorption bases, oil-in-water or water-in-oil emulsions,emulsions carbowax (polyethylene glycols of a variety of molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.

[0063] The quantities of active ingredient necessary for effectivetherapy will depend on many different factors, including means ofadministration, target site, physiological state of the patient, andother medicaments administered. Thus, treatment dosages should betitrated to optimize safety and efficacy. Typically, dosages used invitro may provide useful guidance in the amounts useful for in situadministration of the active ingredients. Animal testing of effectivedoses for treatment of particular disorders will provide furtherpredictive indication of human dosage. Various considerations aredescribed, for example, in Goodman and Oilman's The PharmacologicalBasis of Therapeutics, 7th Edition (1985), MacMillan Publishing Company,New York, and Remington's Pharmaceutical Sciences 18th Edition, (1990)Mack Publishing Co, Easton Perm. Methods for administration arediscussed therein, including oral, intravenous, intraperitoneal,intramuscular, transdermal, nasal, iontophoretic administration, and thelike.

[0064] The compositions of the invention may be administered in avariety of unit dosage forms depending on the method of administration.For example, unit dosage forms suitable for oral administration includesolid dosage forms such as powder, tablets, pills, and capsules, andliquid dosage forms, such as elixirs, syrups, and suspensions. Theactive ingredients may also be administered parenterally in sterileliquid dosage forms. Gelatin capsules contain the active ingredient andas inactive ingredients powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like.

[0065] Examples of additional inactive ingredients that may be added toprovide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

[0066] The concentration of the compositions of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

[0067] The compositions of the invention may also be administered vialiposomes. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. In these preparations the composition of the invention tobe delivered is incorporated as part of a liposome, alone or inconjunction with a molecule which binds to a desired target, such asantibody, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired composition of theinvention of the invention can delivered systemically, or can bedirected to a tissue of interest where the liposomes then deliver theselected therapeutic/immunogenic polypeptide compositions.

[0068] Liposomes for use in the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome size,acid lability and stability of the liposomes in the blood stream. Avariety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al. Ann. Rev. Biophys. Bioeng 9:467 (1980), U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated hereinby reference.

[0069] A liposome suspension containing a composition of the inventionmay be administered intravenously, locally, topically, etc. in a dosewhich varies according to, inter alia the manner of administration, thecomposition of the invention being delivered, and the stage of thedisease being treated.

[0070] For solid compositions, conventional nontoxic solid carriers maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more compositions of the invention of theinvention, and more preferably at a concentration of 25%-75%.

[0071] For aerosol administration, the compositions of the invention arepreferably supplied in finely divided form along with a surfactant andpropellant. Typical percentages of compositions of the invention are0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course,be nontoxic, and preferably soluble in the propellant. Representative ofsuch agents are the esters or partial esters of fatty acids containingfrom 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides maybe employed. The surfactant mayconstitute 0.1%-20% by weight of the composition, preferably 0.25-5%.The balance of the composition is ordinarily propellant. A carrier canalso be included, as desired, as with, e.g., lecithin for intranasaldelivery.

[0072] The constructs of the invention can additionally be delivered ina depot-type system, an encapsulated form, or an implant by techniqueswell-known in the art. Similarly, the constructs can be delivered via apump to a tissue of interest.

[0073] Any of the foregoing formulations may be appropriate intreatments and therapies in accordance with the present invention,provided that the active agent in the formulation is not inactivated bythe formulation and the formulation is physiologically compatible..Polyclonal and/or monoclonal antibodies to the polypeptides of thepresent invention may be prepared. The polypeptides of the inventionthereof may be prepared as described herein, and coupled to a carriermolecule, for example keyhole limpet hemocyanin, and injected intorabbits at selected times over several months. The rabbit sera may betested for immunoreactivity to the polypeptides thereof. Monoclonalantibodies may be made by injecting mice with the polypeptides.Monoclonal antibodies may be screened by methods known in the art, asare described, for example, in Harlow and Lane (1988) Antibodies: Alaboratory manual. Cold Spring Harbor Press, New York, and Goding (1986)Monoclonal antibodies: Principles and Practice (2d ed.) Academic Press,New York. The antibodies will be tested for specific immunoreactivitywith an epitope of the polypeptides. These antibodies will find use indiagnostic assays or as an active ingredient in a pharmaceuticalcomposition.

[0074] For production of polyclonal antibodies, an appropriate targetimmune system is selected, typically a mouse or rabbit, although otherspecies such as goats, sheep, cows, guinea pigs, and rats maybe used.The substantially purified antigen is presented to the immune systemaccording to methods known in the art The immunological response istypically assayed by an immunoassay. Suitable examples include ELISA,RIA, fluorescent assay, or the like. These antibodies will find use indiagnostic assays or as an active ingredient in a pharmaceuticalcomposition.

Rap Nucleic Acid and Proteins

[0075] The present invention also provides a protein (RAP) isolated andpurified from a non-pathogenic Staphylococcus spp.. The RAP protein hasa molecular weight of about 38 kDa. In one embodiment, RAP is theprotein encoded by a polynucleotide comprising the sequence of SEQ IDNO:12, and comprising an amino acid sequence of SEQ ID NO:13. Thesesequences are provided in the Sequence Listing below.

RAP Nucleic Acid

[0076] The term “RAP gene” is used generically to designate RAP genesand their alternate forms. “RAP gene” is also intended to mean the openreading frame encoding specific RAP proteins, and adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation of expression(e.g., promoter region). The gene may be introduced into an appropriatevector for extrachromosomal maintenance or for integration into thehost. In one embodiment the RAP gene comprises the sequence of SEQ IDNO: 12.

[0077] RAP regulatory sequences may be used to identify cis actingsequences required for transcriptional or translational regulation ofRAP expression, especially at different stages of growth (e.g., early,mid, and late log phase), and to identify cis acting sequences and transacting factors that regulate or mediate RAP expression. Suchtranscriptional or translational control regions may be operably linkedto a RAP coding sequence or other coding sequence.

[0078] The nucleic acid compositions used in the subject invention mayencode all or a part of the RAP protein as appropriate. Fragments may beobtained of the DNA sequence by chemically synthesizing oligonucleotidesin accordance with conventional methods, by restriction enzymedigestion, by PCR amplification, etc. For the most part, DNA fragmentswill be of at least about ten contiguous nucleotides, usually at leastabout 15 nt, more usually at least about 18,nt to about 20 nt, moreusually at least about 25 nt to about 50 nt. Such small DNA fragmentsare useful as primers for PCR, hybridization screening, etc. Larger DNAfragments, i.e. greater than 100 nt are useful for production of theencoded polypeptide. For use in amplification reactions, such as PCR, apair of primers will be used.

[0079] The exact composition of the primer sequences is not critical tothe invention, but for most applications the primers will hybridize tothe subject sequence under stringent conditions, as known in the art. Itis preferable to choose a pair of primers that will generate anamplification product of at least about 50 nt, preferably at least about100 nt. Algorithms for the selection of primer sequences are generallyknown, and are available in commercial software packages. Amplificationprimers hybridize to complementary strands of DNA, and will primetowards each other.

[0080] The RAP gene and RAP coding sequence are isolated and obtained insubstantial purity, generally as other than an intact bacterialchromosome. Usually, the DNA will be obtained substantially free ofother nucleic acid sequences that do not include a RAP sequence orfragment thereof, generally being at least about 50%, usually at leastabout 90% pure and are typically “recombinant”, i.e. flanked by one ormore nucleotides with which it is not normally associated on a naturallyoccurring chromosome.

[0081] The DNA sequences are used in a variety of ways. They may be usedas probes for identifying RAP coding sequences of other strains ofStaphylococcus or of other bacteria. Homologs isolated from otherstains, species, or genera generally have substantial sequencesimilarity to one another, i.e. at least 75%, usually at least 90%, moreusually at least 95% sequence identity. In general, RAP-encodingsequences of the invention (including homologs, variants, etc.) arecharacterized by having a sequence identity greater than at least about65%, preferably at least about 75%, more preferably at least about 85%,and can be greater than at least about 90% or more as determined by theSmith-Waterman homology search algorithm as implemented in MPSRCHprogram (Oxford Molecular). For the purposes of this invention, asequence identity is calculated using the Smith-Waterman algorithm asfollows: Global DNA sequence identity must be greater than 65% asdetermined by the Smith-Waterman homology search algorithm asimplemented in MPSRCH program (Oxford Molecular) using an affine gapsearch with the following search parameters: gap open penalty, 12; andgap extension penalty, 1.

[0082] Nucleic acids having sequence similarity can also be detected byhybridization under low stringency conditions, for example, at 50° C.and 6×SSC (0.9 M saline/0.09 M sodium citrate) and remain bound whensubjected to washing at 55° C. in 1×SSC (0.15 M sodium chloride/0.015 Msodium citrate). In addition, sequence identity may also be determinedby hybridization under high stringency conditions, for example, at 50°C. or higher and 0.1×SSC (15 mM saline/0.15 mM sodium citrate). By usingprobes, particularly labeled probes of DNA sequences, one can isolatehomologous or related genes. It may also be possible to identifyhomologs of RAP from mammalian sources.

[0083] The RAP-encoding DNA may also be used to detect expression of thegene in a biological specimen. Methods and materials for probing asample for the presence of particular nucleotide sequences are wellestablished in the literature and do not require elaboration here. mRNAis isolated from a cell sample. mRNA may be amplified by RT-PCR, usingreverse transcriptase to form a complementary DNA strand, followed bypolymerase chain reaction amplification using primers specific for thesubject DNA sequences. Alternatively, mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to an RAP sequenceis indicative of RAP gene expression in the sample.

[0084] The RAP nucleic acid sequence may be modified for a number ofpurposes, particularly where they will be used intracellularly, forexample, by being joined to a nucleic acid cleaving agent, e.g. achelated metal ion, such as iron or chromium for cleavage of the gene;or the like.

[0085] The RAP coding sequence and/or promoter sequence may be mutatedin various ways known in the art to generate targeted changes inpromoter strength, sequence of the encoded protein, etc. The DNAsequence or product of such a mutation will be substantially similar tothe sequences provided herein, i.e. will differ by at least onenucleotide or amino acid, respectively, and may differ by at least twobut not more than about ten nucleotides or amino acids. The sequencechanges may be substitutions, insertions or deletions. Deletions mayfurther include larger changes, such as deletions of a domain. Othermodifications of interest include production of fusion proteins (e.g.,with green fluorescent proteins (GFP), luciferase, and the like).

[0086] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for scanning mutations may be found in Gustin etal., 1993 Biotechniques 14:22; Barany, 1985 Gene 37:111-23; Colicelli etal., 1985 Mol Gen Genet 199:537-9; and Prentki et al., 1984 Gene29:303-13. Methods for site specific mutagenesis can be found inSambrook et al., 1989 Molecular Cloning: A Laboratory Manual, CSH Press,pp. 15.3-15.108; Weiner et al., 1993 Gene 126:35-41; Sayers et al., 1992Biotechniques 13:592-6; Jones and Winistorfer, 1992 Biotechniques12:528-30; Barton et al., 1990 Nucleic Acids Res 18:7349-55; Marotti andTomich, 1989 Gene Anal Tech 6:67-70; and Zhu 1989 Anal Biochem177:120-4.

RAP Protein

[0087] RAP protein can be produced by any suitable means, e.g., byisolated from a bacteria that naturally expresses RAP, by recombinantmeans (e.g., by expression of a polynucleotide having a sequence of SEQID NO:12), by synthetic means, and the like.

[0088] In one embodiment, RAP is isolated directly from a strain ofStaphylococcus producing RAP, e.g., S. aureus. Typically, wild typecells are collected from postexponential culture broth. Cells are thencentrifuged and the supernatant subjected to purification by, forexample, filtration followed by lyophilizetion, resuspensionin water,and further purification.

[0089] The staphylococci bacterium from which RAP may be isolated mayinclude, but is not necessarily limited to, S. aureus, S. capitus, S.wameri, S. capitis, S. caprae, S. carnosus, S. saprophyticus, S.chronii, S. simulans, S. caseolyticus, S. epidermidis, S. haemolyticus,S. hominis, S. hyicus, S. kloosii, S. lentus, S. lugdunensis, S. scruri,S. simulans, and S. xylosus. Preferably RAP is isolated from S. aureus.

[0090] In another embodiment, RAP-encoding nucleic acid is employed tosynthesize full-length RAP protein or fragments thereof, particularlyfragments corresponding to functional domains (e.g., phosphorylationsites that interact with RAP, etc.); and including fusions of thesubject polypeptides to other proteins or parts thereof. For expression,an expression cassette may be employed, providing for a transcriptionaland translational initiation region, which may be inducible orconstitutive, where the coding region is operably linked under thetranscriptional control of the transcriptional initiation region, and atranscriptional and translational termination region. Varioustranscriptional initiation regions may be employed that are functionalin the expression host.

[0091] The polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. Alternatively, RAPfragments can be synthesized.

[0092] With the availability of the polypeptides in large amounts, byemploying an expression host, RAP protein can be isolated and purifiedin accordance with conventional ways, e.g., using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. The purified protein will generally be at leastabout 80% pure, preferably at least about 90% pure, and may be up to andincluding 100% pure.

[0093] The RAP proteins can be used for the production of antibodies,where short fragments provide for antibodies specific for the particularpolypeptide, and larger fragments or the entire protein allow for theproduction of antibodies over the surface of the polypeptide. Antibodiesmay be raised to the wild-type or variant forms of RAP. Antibodies maybe raised to isolated peptides corresponding to these domains, or to thenative protein, e.g. by immunization with cells expressing RAP,immunization with liposomes having RAP protein inserted in the membrane,etc.

Anti-RAP Antibodies

[0094] The present invention also provides an antibody that specificallybinds and is immunoreactive with RAP. The antibody may be monoclonal,polyclonal or humanized, and is prepared using methods well known in theart. In general, antibodies are prepared in accordance with conventionalways, where the protein or an antigenic portion thereof is used as animmunogen, by itself or conjugated to known immunogenic carriers, e.g.KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like.Various adjuvants may be employed, with a series of injections, asappropriate. For monoclonal antibodies, after one or more boosterinjections, the spleen is isolated, the lymphocytes immortalized by cellfusion, and then screened for high affinity antibody binding. In apreferred embodiment, the spleen or lymph node cells and myeloma cellsare mixed in about 20:1 to about 1:1 ratio, but preferably in about 2:1ratio. It is preferred that the same species of animal serve as thesource of somatic and myeloma cells used in the fusion procedure, wherethe animal is chosen from rat, mouse, rabbit, cow, chicken, turkey, orman. The fusion of the somatic and myeloma cells produces a hybridoma,which is grown in culture to produce the desired monoclonal antibody bystandard procedures. For further description, see, for example,Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., ColdSpring Harbor Laboratories, Cold Spring Harbor, New York, 1988. Ifdesired, the mRNA encoding the heavy and light chains may be isolatedand mutagenized by cloning in E. coli, and the heavy and light chainsmixed to further enhance the affinity of the antibody. Alternatives toin vivo immunization as a method of raising antibodies include bindingto phage “display” libraries, usually in conjunction with in vitroaffinity maturation.

[0095] The polyclonal antibodies of the present invention may beproduced by injecting a rat, a mouse, a rabbit, a cow, a chicken, or aturkey with RAP to initiate an immunogenic response. RAP may be coupledto a protein carrier such as deyhole limpet hemocyanin (KLH) or bovineserum albumin (BSA). An adjuvant may also be used. After a suitableamount of time to establish a high-titer of anti-RAP antibodies, theserum or eggs are collected. The presence of antibody in the serum oreggs may be tested by radioimmunoassay (RIA), by enzyme-linkedimmunosorbent assay (ELISA), or by immunoprecipitation. Theimmunoglobulins may be isolated by the sequential precipitation methods,by conventional methods of “salting out” the protein fractions from asalt solution, or by chromatographical methods well known to thoseskilled in the art.

Identifying Agents Suitable for Treating Staphylococcus Infection

[0096] Of particular interest in the present invention is theidentification of agents that have activity in affecting the expressionand/or function of RAP. In general agents of interest are those thatinhibit RAP activity, e.g., by inhibiting the ability of RAP to effectactivation of rnaiii. Such agents are candidates for development oftreatments for infection of pathogenic Staphylococcus. Of particularinterest are screening assays for agents that have a low toxicity forhuman cells and/or high specificity for Staphylococcus, preferably withsubstantially no or little pressure for selection of strains resistantto the action of the agent, and without substantially affecting normalflora of the host (e.g., as distinguished from wide-spectrumantibiotics).

[0097] The term “agent” as used herein describes any molecule, e.g.protein or pharmaceutical, with the capability of altering RAP activity,or mimicking or enhancing RIP activity, as described above. Generally aplurality of assay mixtures are run in parallel with different agentconcentrations to detect differential responses to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e. at zero concentration or below the level ofdetection.

[0098] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including, but not limited to: peptides,saccharides, fatty acids, steroids, pheromones, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

[0099] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial (e.g., non-pathogenicStaphylococcus), fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means, and may be used to producecombinatorial libraries. Known pharmacological agents may be subjectedto directed or random chemical modifications, such as acylation,alkylation, esterification, amidification, etc. to produce structuralanalogs.

Screening of Candidate Agents

[0100] A wide variety of in vitro assays may be used to screen candidateagents, including labeled in vitro binding assays, e.g., protein-proteinbinding, protein-DNA binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, and the like. Purifiednaturally-occurring or recombinant RAP and RIP proteins, and/orsynthetically produced peptides or fragments of RAP and/or RIP, can beused in various screening assays to identify ligands or substrates thatbind to, modulate (e.g., increase or inhibit), or mimic the action ofthe native proteins. The purified proteins may also be used fordetermination of three-dimensional crystal structure, which can be usedfor modeling intermolecular interactions, transcriptional regulation,etc.

[0101] The screening assay can be a binding assay, wherein one or moreof the molecules may be joined to a label, and the label directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures. In general,the particular type of screening assay employed will preferably oneamenable to parallel, simultaneous screening of a large number ofcandidate agents.

[0102] Screening assays of the present invention encompass assays thatexamine the effect of candidate agents on the roles of RAP, and RIP inRNAIII production and/or virulence factor production. For example, thecandidate agent may be contacted with pathogenic Staphylococcus and thelevels of rnaiii transcription in the presence of the agent compared tornaiii transcription levels in the presence of RIP, RAP, and/or acombination of RIP and RAP. Such screening assays can utilizerecombinant host cells containing reporter gene systems such as CAT(chloramphenicol acetyltransferase), β-galactosidase, and the likeoperably associated with rnaiii or virulence factor genes to facilitatedetection of rnaiii or virulence gene transcription or to facilitatedetection of RNAIII or virulence factor production. Alternatively, thescreening assay can detect rnaiii or virulence factor transcriptionusing hybridization techniques (e.g., Northern blot, PCR, etc.) wellknown in the art.

[0103] A variety of other reagents may be included in the screeningassays described herein. Where the assay is a binding assay, theseinclude reagents like salts, neutral proteins, e.g. albumin, detergents,etc. that are used to facilitate optimal protein-protein binding,protein-DNA binding, and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, antimicrobial agents, etc. maybe used. The mixture of components are added in any order that providesfor the requisite binding. Incubations are performed at any suitabletemperature, typically between 4° C. and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Screening of Candidate Agents in an Animal Model

[0104] Agents having a desired activity as determined in the assaysdescribed above can be further screened for their ability to affectStaphylococcus virulence factor production, and to affect Staphylococcusinfection, in a non-human animal model. The animal model selected willvary with a number of factors including, but not limited to, theparticular pathogenic strain of Staphylococcus against which candidateagents are to be screened, the ultimate host for which the candidateagents are to serve as therapeutics, etc. Animals suitable for use inscreening assays include any animal susceptible to infection by theselected Staphylococcus species. For example, where the Staphylococcusspecies is S. aureus, the animal model can be a rodent model, preferablya mouse model.

[0105] In general, the candidate agent is administered to a non-humananimal susceptible to Staphylococcus infection, where the animal hasbeen previously infected with Staphylococcus or receives an infectiousdoes of Staphylococcus in conjunction with the candidate agent.Preferably, the animal has no detectable RAP antibodies. The candidateagent can be administered in any manner desired and/or appropriate fordelivery of the agent in order to effect a desired result. For example,the candidate agent can be administered by injection (e.g., by injectionintravenously, intramuscularly, subcutaneously, or directly into thetissue in which the desired affect is to be achieved), topically,orally, or by any other desirable means. Normally, this screen willinvolve a number of animals receiving varying amounts and concentrationsof the candidate agent (from no agent to an amount of agent hatapproaches an upper limit of the amount that can be deliveredsuccessfully to the animal), and may include delivery of the agent indifferent formulations. The agents can be administered singly or can becombined in combinations of two or more, especially where administrationof a combination of agents may result in a synergistic effect.

[0106] The effect of agent administration upon the animal model can bemonitored by any suitable method, such as assessing the number and sizeof Staphylococcus-associated lesions, overall health, etc. Where thecandidate agent affects Staphylococcus infection in a desirable manner(e.g., by reducing infectious load, facilitating lesion regression,etc.), the candidate agent is identified as an agent suitable for use intreatment of Staphylococcus infection.

Identified Candidate Agents

[0107] The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of pathogenic Staphylococcus infection. The therapeutic agentsmay be administered in a variety of ways, orally, topically,parenterally e.g. subcutaneously, intraperitoneally, intravascularly,intrapulmonary (inhalation), etc. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %.

[0108] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Treating Staphylococcus Infection

[0109] The invention provides a method for preventing or treating ahuman or an animal susceptible to infection by a pathogenicStaphylococcus (e.g., S. aureus in humans) by administering an agentthat inhibits RAP activity in facilitating virulence factor production,e.g., by inhibition RAP-mediated activiation of RNAIII and subsequentvirulence factor production.

[0110] In one embodiment, the host is treated by administration of RIPor with a RAP inhibitor, such as an anti-RAP antibody, or both. In oneembodiment the RAP inhibitor is co-administered with other RAPinhibitors and/or co-administered with other inhibitors of S. aureusvirulence factor production, e.g., co-administered with RIP. In anotherembodiment a RAP inhibitor, RIP, and a RAP inhibitor (e.g., an anti-RAPantibody) are administered. Such combination therapies (e.g.,administration of multiple RAP inhibitory agents; administration of RAPand RIP; and/or administration of RAP inhibitor, RIP, and/or RAPinhibitor) may involve co-administration or sequential administration ofthe active components. The dosage regimen may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the therapeutic situation. The active compounds may beadministered in any convenient manner, such as by oral, intravenous,intramuscular, subcutaneous, buccal, transdermal, or inhalation routes.

[0111] Formulations composed of RIP, RAP inhibitor, or both areadministered at a therapeutically effective dosage, e.g., a dosagesufficient to improve the chance of successful prevention or treatmentof infection. Administration of such a formulation can be via any of theaccepted modes of administration for agents that serve similarutilities, preferably by systemic administration.

[0112] Human dosage levels for treating infections are known andgenerally include a daily dose from about 0.1 to 500.0 mg/kg of bodyweight per day, preferably about 6.0 to 200.0 mg/kg, and most preferablyabout 12.0 to 100.0 mg/kg. Generally, it is sought to obtain a serumconcentration of such a formulation approximating or greater thancirculating levels needed to reduce or eliminate any infection in lessthan 10 days. For administration to a 70 kg person, the dosage rangewould be about 50 mg to 3.5 g per day, preferably about 100 mg to 2 gper day, and most preferably about 200 mg to 1 g per day. The amount offormulation administered will, of course, be dependent on the subjectand the severity of the affliction, the manner and schedule ofadministration and the judgment of the prescribing physician.

[0113] In employing formulation for treatment of infections, anypharmaceutically acceptable mode of administration can be used. Theformulations can be administered either alone or in combination withother pharmaceutically acceptable excipients, including solid,semi-solid, liquid or aerosol dosage forms, such as, for example,tablets, capsules, powders, liquids, gels, suspensions, suppositories,aerosols or the like. The formulations can also be administered insustained or controlled release dosage forms (e.g., employing a slowrelease bioerodable delivery system), including depot injections,osmotic pumps, pills, transdermal and transcutaneous (includingelectrotransport) patches, and the like, for prolonged administration ofa predetermined rate, preferably in unit dosage forms suitable forsingle administration of precise dosages.

[0114] The compositions will typically include a conventionalpharmaceutical carrier or excipient and a formulation of the invention.In addition, these compositions may include other active agents,carriers, adjuvants, etc. Generally, depending on the intended mode ofadministration, the pharmaceutically acceptable composition will containabout 0.1% to 90%, preferably about 0.5% to 50%, by weight of activecompound, the remainder being suitable pharmaceutical excipients,carriers, etc. Actual methods of preparing such dosage forms are known,or will be apparent, to those skilled in this art. For example, seeRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 15th Edition, 1975.

[0115] Parental administration is generally characterized by injection,either subcutaneously, intradermally, intramuscularly, or intravenously,preferably subcutaneously. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol or the like. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, solubility enhancers, and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, cyclodextrins, and the like.

[0116] The percentage of active ingredient contained in such parentalcompositions is highly dependent on the specific nature thereof, as wellas the needs of the subject. However, percentages of active ingredientof 0.01% to 10% in solution are employable, and will be higher if thecomposition is a solid which will be subsequently diluted to the abovepercentages. Preferably, the composition will comprise 0.2-2% of theactive ingredient in solution.

[0117] A more recently devised approach for parental administrationemploys the implantation of a slow-release or sustained-release system,such that a constant level of dosage is maintained. Various matrices(e.g., polymers, hydrophilic gels, and the like) for controlling thesustained release, and for progressively diminishing the rate of releaseof active agents are known in the art. See U.S. Pat. Nos. 3,845,770(describing elementary osmotic pumps); U.S. Pat. Nos. 3,995,651,4,034,756 and 4,111,202 (describing miniature osmotic pumps); U.S. Pat.Nos. 4,320,759 and 4,449,983 (describing multichamber osmotic systemsreferred to as push-pull and push-melt osmotic pumps); and U.S. Pat. No.5,023,088 (describing osmotic pumps patterned for the sequentially timeddispensing of various dosage units).

[0118] Formulations of active components may also be administered to therespiratory tract as a nasal or pulmonary inhalation aerosol or solutionfor a nebulizer, or as a microfine powder for inhalation, alone or incombination with an inert carrier such as lactose, or with otherpharmaceutically acceptable excipients. In such a case, the particles ofthe formulation may advantageously have diameters of less than 50microns, preferably less than 10 microns. See, e.g., U.S. Pat. No.5,364,838, which discloses a method of administration for insulin thatcan be adapted for the administration of formulations of the presentinvention.

Vaccination

[0119] The invention provides a vaccine for inoculating a human or aanimal susceptible to infection by a pathogenic Staphylococcus (e.g., S.aureus) by administering RAP, or an antigenically effective portion ofRAP, in a pharmaceutically acceptable carrier, which may optionallycomprise an adjuvant. Formulations appropriate for elicitation of theimmune response are well known in the art. In general, the host isexposed to the antigen, such as RAP, which perturbs the host's immunesystem and results in an immune response towards the antigen. Anadjuvant can be added with the antigen to increase the immune responseto the antigen. The amount of polypeptide administered is an amountsufficient to elicit a protective immune response in the host. Methodsfor determining such appropriate amounts are routine and well known inthe art. For example, RAP and/or antigenically effective portion(s)thereof can be used to vaccinate an animal model of Staphylococcusinfection. The amounts effective in such animal models can beextrapolated to other hosts (e.g., livestock, humans, etc.) in order toprovide for an amount effective for vaccination.

Coated Devices

[0120] The invention provides for a device, the surface of which iscoated with a composition having an amount of a RAP inhibitory agent(e.g., an anti-RAP antibody or RIP peptides) effective to inhibitproduction of virulence factors by pathogenic Staphylococcus. The coateddevice may be any device which may be associated with a risk ofStaphylococcus infection or exposure of a host (e.g., surgery patient,menstruating female, etc.) to Staphylococcus virulence factors.

[0121] Coated devices encompassed by the present invention include, butare not limited to, catheters, needles, surgical instruments (e.g.,scalpels, sponges, retractors, etc.), bandages and bandage materials(e.g., gauze, dressings, etc.), artificial joints, heart valves, andtampons. Such devices have a tendency to bring Staphylococci intocontact with the host, or to attract colonizations by staph bacteria(e.g., tampons). In such situations, the coated devices may prevent orreduce Staphylococcus infection, or prevent or reduce the development ofserious symptoms associated with exposure to Staphylococcus virulencefactors.

[0122] The following examples are intended to illustrate, not limit thescope of this invention

EXAMPLES I. In Vivo Studies in Mice A. Purification of RAP

[0123] In these experiments RAP was purified from post exponentialsupernatants of wild-type S. aureus (FIG. 1A) as described by Balaban etal. (Proc. Natl. Acad. Sci. U.S.A. 92, 1619 (1995)). Peak fractions(FIGS. 1B, 1C) contained a protein of about 38 kD (FIG. ID).HPLC-purified RAP was run on SDS PAGE, blotted onto a PVDF membrane,Coomassie stained, and the purified 38-kD protein used for N-terminalsequencing by Edman degradation. The NH₂-terminal sequence of RAP wasdetermined to be IKKYKPITN (SEQ ID NO:6). This sequence showed nosignificant homology to known proteins. We also purified RAP from postexponential supernatants of an agr-null strain in which the entire agrlocus was replaced with the tetM marker (Novick et al. EMBO J. 12:3967(1993)), which suggests that RAP is independent of agr.

B. Immunization with RAP

[0124] To test whether immunization with RAP can inhibit S. aureusinfection, we used the murine model of cutaneous infection (Bunce etal., Infect. Immun.60, 2636 (1992)). In this model, when the wild-typeSmith diffuse (SD) strain of S. aureus is injected subcutaneouslytogether with dextran beads (Cytodex), a visible, measurable lesion(cellulitis) is induced after 24 hrs. In contrast, no lesion is inducedin animals injected with cytodexbeads alone.

[0125] Four-eight week old (20-30 g), outbred, immunocompetent, hairlessmale mice, strain Crl:SKHl(hrhr)Br were obtained from Charles River,Wilmington, Mass. and used for these experiments. For prophylacticvaccination with RAP (purified from a wild type S. aureus strain) orRAP* ( purified from an agr-null), 10 μg RAP, purified on a gelfiltration column as described by Balaban et al. (supra), was injectedtogether with complete Freund's adjuvant (CFA) on first injection andincomplete Freund's adjuvant (1CFA) on second and third injectionsubcutaneously, into 4 week old male hairless immunocompetent mice ondays 0, 7 and 21. Control mice were either injected with the adjuvantalone or not injected at all (untreated). Vaccinated and control micewere challenged on day 31 with 1.24×10⁸ Smith Diffuse S. aureussubcutaneously together with 1 mg cytodex beads (10) to induce a localinfection. The size of the lesion was measured daily and presented asarea=0.5 {p(length)(width)} (Table 1).

[0126] After challenge with S. aureus, 72% ({fraction (24/33)})RAP-vaccinated mice remained free of disease as compared to 30%({fraction (3/10)}) controls immunized with complete Freund's adjuvant(CFA) and 0% of untreated controls. This difference was statisticallysignificant (RAP vs untreated: p<0.0001; RAP vs CFA: p<0.003 1) usingFisher's exact probability test Fisher's Exact Probability Test was usedto compare proportions of mice developing lesions and mice developinganti-RAP antibodies among the experimental groups (RAP-vaccinated, CFAcontrols, untreated controls). Among animals developing lesions postchallenge with S. aureus, the size of the lesions was compared usingsingle factor Analysis of Variance. Post-hoc testing was performed usingFisher's protected least significant difference.

[0127] In addition, in mice that developed lesions, the mean lesion sizewas 50% smaller than that of CFA control mice (177 mm²) and 76% smallerthan untreated controls (370 mm²). Animals that died had extensivelesions which spread to over one quarter of their body size. Only 3%({fraction (1/33)}) of RAP-vaccinated animals died as a result ofchallenge whereas 22% of control animals died ({fraction (5/22)}).

[0128] To determine if antibodies to RAP were generated, sera fromvaccinated and control animals were analyzed by immunoblotting (FIG. 2A)and binding activity to wild-type S. aureus post-exponentialsupernatants that contained RAP. To estimate antibody levels against theinjected antigen, a drop of blood (50 μl) was collected from the tip ofthe tail before vaccination (pre-immune sera) and 7 days after the thirdvaccination period (post-immune sera, 3 days before bacterialchallenge). Anti-RAP antibody titer was determined by western blotting.Sera was added to the blot (containing postexponential supernatants) inincreasing dilutions, until no band appeared. The highest dilution whichstill reacted with RAP was the determined titer. Animals vaccinated withRAP developed antibodies to a 38-kD protein, and could also bind topurified RAP.

[0129] Most ({fraction (33/34)}) vaccinated animals developed antibodiesto RAP (with titers ranging from 1:50-1:16,000) as a result of theinjections, whereas control animals did not. However, some of theanimals contained pre-immune antibodies to RAP (titers ranging from1:40-1:2000) and developed smaller lesions upon challenge with S.aureus. The titer of antibodies to RAP (in a total of 81 vaccinated orcontrol animals) inversely correlated with lesion size (FIG. 2B). Forpurposes of this calculation, animals that died (of extensivecellulitis) were assumed to have a lesion size of 981 mm².

[0130] Mice vaccinated with RAP which purified from a S. aureus agr-nullstrain as described above had the same degree of protection as animalsvaccinated with RAP which was purified from a wild-type strain. Thisrules out contribution of products from other genes known to regulateRNAIII, such as the octapeptide encoded by agrD (Guangyong et al. Proc.Natl. Acad. Sci. USA. 92, 12055 (1995)). TABLE 1 Vaccination with RAP asan antigen Treatment no lesion lesion death group (total n) n (%) n meansize (mm²) n (%) RAP (24) 17 (71) 7 (96) 1 (4) RAP* (9) 7 (78) 2 (84) 0(0) CFA (10) 3 (30) 10 (177) 2 (20) Untreated (12) 0 (0) 12 (370) 3 (25)

C. Purification and Amino Acid Sequence of RIP

[0131] The non pathogenic coagulase negative staphylococcus presumed tobe S. xylosus (ATCC 55619) produces the peptide RIP which inhibitsRNAIII transcription (FIG. 3) and competes with RAP for the activationof virulence (Balaban et al, supra). HPLC-purified REP (ibid) wassubmitted to Edman degradation sequencing. The amino acid sequence wasdetermined to be YSPXTNF (SEQ ID NOS:3 and 4), where X is a modifiedamino acid, C, I, or W, which has sequence homology to the NH₂-terminalsequence of RAP. Without being limited to any one theory, this suggeststhat gene encoding RIP may be a derivative of the gene encoding RAP.

[0132] A synthetic peptide having the sequence YSPWTNF ((SEQ ID NO:7)denoted Pep herein) was synthesized and tested for its ability toinhibit RNAIII in vitro. The synthetic peptide inhibited induction ofRNAIII comparably to RIP (FIG. 3).

D. Suppression of Infection by Purified RAP and RIP

[0133] Purified (Balaban etal, Proc. Natl. Acad. Sci. U.S.A. 92: 1619(1995)) and synthetic RIP were tested for their ability to suppressinfection in the murine cutaneous S.aureus infection model. SmithDiffuse S. aureus (8.5×10⁷−1.4×10⁹) were incubated in the presence ofRIP which was purified from 5 ml postexponential culture broth of ATCC55619 in saline, or with saline only as a control, with 0.5 mg syntheticRIP (Pep) in a final DMSO (in saline) solution of 3%, or only with 3%DMSO in saline as a control, for 30 min at 37° C. The bacteria+RIP,bacteria+Pep, bacteria+saline or bacteria+DMSO mixture was injectedsubcutaneously together with cytodex beads (1 mg) into 8 week old malehairless immunocompetent mice to induce a local infection. The size ofthe lesion was measured daily. For these experiments mice werepre-screened to eliminate individuals with anti-RAP antibodies. A fixedamount of RIP (about 10 mg) attenuated infections caused by increasinginocula of the Smith Diffuse (SD) strain of S. aureus.

[0134] Of the animals that were injected with 8.5×10⁷ bacteria togetherwith RIP, three of four developed no infection at all, as compared toonly one of four control animals that were injected with the bacteriaand saline (Table 2). When an increased inoculum of bacteria was used(1.4×10⁵ cells per injection), four of eight animals were protected,whereas the remaining four developed a lesion that was 55% smaller thanthat of control animals (Table 2). All ({fraction (7/7)}) of the controlanimals challenged with SD and saline developed a lesion. When a highernumber of bacteria was used (1.4×10⁹), the synthetic RIP (0.5 mg Pep)protected animals, where 90% ({fraction (9/10)}) of the animals showedno sign of disease (Table 2). TABLE 2 Vaccination or suppression of S.aureus SD infections no lesion lesion death Treatment (total n) n (%) nmean size (mm²) n (%) RIP Suppression of 8.5 × 10⁷ SD SD + RIP (4) 3(75) 1 (33) 0 (0) SD + Saline (4) 1 (25) 3 (39) 0 (0) RIP Suppression of1.4 × 10⁸ SD SD + RIP (8) 4 (50) 4 (45) 0 (0) SD + Saline (6) 0 (0) 6(100) 0 (0) RIP and Pep Suppression of 1.4 × 10⁹ SD SD + RIP (10) 3 (30)0 (0) 4 (40) SD + Saline (10) 2 (20) 6 (160) 2 (20) SD + Pep (10) 9 (90)1 (56) 0 (0) SD + DMSO (9) 2 (20) 4 (128) 3 (22)

E. Nucleotide Sequence of RIP

[0135] Degenerate oligonucleotides were designed from the amino acidsequence YSPWTNF (SEQ ID NO:7). The rip gene was amplified by PCR usingTaq DNA polymerase. The PCR product was cloned into pCR2.1 (Invitrogen)and sequenced. The DNA sequence of the tip gene was determined to be TATTCG CCG TGG ACC AAT TTT (SEQ ID NO:5).

F. Comparison of Synthetic and Natural RIP Peptides

[0136] Synthetic peptides corresponding to RIP were synthesized andtested for their ability to inhibit RNAIII in vitro (FIGS. 4A,B) or tocompete with RAP on the activation of RNAIII (FIG. 4C). In those FiguresRAP includes the sequence IKKYKPITN (SEQ ID NO:6); Pep 1 is PCTNF (SEQID NO:8); Pep2 is YSPWTNF (SEQ ID NO:7); B1 is YKPITNF (SEQ ID NO:9); B2is YSPYINF (SEQ ID NO:10); and B3 is YKPWTNF (SEQ ID NO:11).

[0137] In these experiments RIP peptides or control buffer were added to2×10⁷ ATCC 55620 cells containing the agr P3-blaZ fusion plasmid. Cellswere grown in a microtiter plate from early exponential phase of growth(before rnaiii is normally activated) for 2 hr (when rnaiii is normallyactivated) at 37° C. with shaking. The reaction was stopped by theaddition of 10 μl CY containing azide. β-lactamase activity was measuredby the addition of 50 μL nitrocefin (132 μg/ml in 0.1 M sodium phosphatebuffer, pH 5.8) and read at 490-650 nm. To determine the competitionbetween RAP and purified or synthetic RIP, RAP was added to cells in thepresence or absence of RIP and RNAIII was measured 2 hr later. Insummary, Pep2 inhibited RNAIII 100% at less than 1 ng. B1 inhibitedRNAEI 50% at 2 μg; B2 inhibited RNAIII 50% at 66.6 μg; and B3 inhibitedRNAIII 100% at 4 μg.

G. Discussion

[0138] Regulatory mechanisms involving autoinducers have been describedfor other bacterial systems (Rappuoli, etal. in: Signal Transduction andBacterial Virulence, R. Rappuoli, V. Scarlato, B. Arico eds. (SpringerVerlag, Heidelberg 1995). pp.1-4.), including competence and sporulationin Bacillus subtilis and in Streptococcus pneumoniae (Magnuson et al.Cell 77, 207 (1994)), conjugation in Enterococcus faecalis (Swift etal., Trends in Microbiol. 2, 193 (1994)) and elastase production inPseudomonas aeruginosa (Pearson et al. Proc. Natl. Acad. Sci. U.S.A. 92,1490 (1995)). Furthermore, compounds have been identified which inhibitthe phosphorylation of the bacterial two component signal transductionsystem in P. aeruginosa (Roychoudhury, etal., Proc. Natl. Acad. Sci.U.S.A. 90, 965 (1993)). Targeting the autoinducers of virulence or thesignal transduction they activate may therefore be useful in preventingpathogenesis of other bacteria known to be regulated by global regulons.

[0139] Current bacterial vaccines target S. aureus or exotoxins itproduces ((Lee, Trends in Microbiol. 4,162 (1996)), R. Naso and A.Fattom, in: Novel Strategies in Design and Production of Vaccines, S.Cohen and A. Shafferman, eds. (Plenum Press, New York, 1996) pp.133-140), but these approaches met with limited success. With aninexorable increase in antibiotic resistance among bacteria in general(Arthur et al., Antimicrob. Agents and Chemother. 37, 1563 (1993)) andamong staphylococci in particular (Noble et al. FEMS Microbiol. Lett.93, 195 (1992)), there is a need to develop new methods to controlbacterial infections. Our approach is to interfere directly withbacterial virulence by interfering with the signal transduction thatleads to the production of toxins. By reducing the pathogenic potentialof the bacteria, this approach would be synergistic with currentanti-microbial therapies and natural host immune mechanisms. Becausedirected suppression of virulence would not kill the bacteria but ratherinterfere with its pathogenicity, there would likely be a decrease inselective pressures for the emergence of new resistant S. aureisstrains.

II. Analysis of Cows for Anti-RAP Antibodies

[0140] Serum was collected from lactating dairy cows with one or morepositive milk cultures for S. aureus (positive) and from lactating cowsthat have no record of having clinical case of S. aureus mastitisthrough one lactation (negative) (Table 3 A) and from calves which are 1month and 4 months old (Table 3B). Sera were tested for anti-RAPantibodies by western blotting against post exponential supernatants ofwild type S. aureus-containing RAP. As shown in Table 3 A, only 10%({fraction (2/20)}) of S. aureus-positive cows contain anti-RAPantibodies, while 63% ({fraction (7/11)}) of S. aureus-negative cowscontain anti-RAP antibodies. As shown in Table 3B, 38-46% of the calvescontained anti-RAP antibodies. S. aureus-negative cows as well as calveswill be followed in the future for correlation between titer of anti-RAPantibodies and natural infection rates. As also shown in Table 3 A, 60%({fraction (12/20)}) of the positive cows as compared to 18% ({fraction(2/11)}) of the negative cows also contained antibodies to variousunidentified proteins in S. aureus supernatant (presumed to beantibodies to other S. aureus proteins).

[0141] Thus, these data indicate that a majority of dairy cows that arenegative for S. aureus mastitis naturally contain anti-RAP antibodies.These results support the use of RAP as a useful vaccine target site forthe prevention of staphylococcal infections. TABLE 2A Anti-RAPAntibodies in Cows other S. aureus Cow n RAP proteins no anti-S. aureusnegative 11 7(63%)  2(18%) 1(18%) positive20 2(10%) 12(60%) 6(30%)

[0142] TABLE 2B Anti-RAP Antibodies in Calves Calves n RAP other S.aureus proteins no anti-S. aureus 1 month old 20 5(38%) 2(10%) 13(65%) 4months old 20 6(46)  1(5)  13(65%)

III. Preparation of Recombinant RAP (Antigen) A. Purification of RAP andN Terminal Sequencing

[0143] RAP is continuously produced by S. aureus and can be purifiedfrom postexponential culture broth. To purify RAP, wild-type S. aureusRN6390B or agr null RN6911 cells were grown to the postexponential phaseof growth. Growth culture was centrifuged at 6000×g for 10 min at 4° C.The supernatant was collected and filtered through a 0.22-μm filter toremove residual cells. The supernatant was lyophilized and resuspendedin water to {fraction (1/10)} of the original volume (total 10×).

[0144] Fifteen milliliters of total 10× was applied to a 10-kD cutoffmembrane (Centriprep 10 (Amicon)). This enabled us to concentrate thematerial further and to remove material smaller than 10 kDa. Onemilliliter concentrated material greater than 10 kDa was washed twice inphosphate buffered saline (PBS) by resuspending it each time in 15-mlPBS and reconcentrating it on the Centriprep 10, and the materialgreater than 10 kDa collected (>10). One hundred microliter materialgreater than 10 kDa was applied to an HPLC gel filtration column(Bio-Sil SEC-125 300×7.8 mm, Bio-Rad) in 1 mM PBS, pH 7.2 (0.1×PBS), ata flow rate of 0.5 ml/min, and 1-ml fractions collected. Fractions wereconcentrated to {fraction (1/10)} of their original volume bylyophilization and tested for activation of RNAIII synthesis asdescribed below.

[0145] Active gel filtration fraction (1 ml) was fractionated by anionexchange chromatography (HPLC SynchroPak Q300, Keystone Scientific,Inc.) in water, pH 7.2. The fraction that activated RNAIII synthesis(eluted at 0.75M NaCl) was collected, separated by SDS-PAGE, and westernblotted. PVDF membrane was stained by coomassie, and protein band ofapproximately 38 kDa was amino acid-sequenced commercially by Edmandegradation chemistry.

[0146] The NH2-terminal sequence of RAP was determined to be IKKYKPITN(SEQ ID NO:6). This sequence was compared to the S. aureus database, andthe sequence of the open reading frame suggests that it is a possible279-amino acid polypeptide (FIG. 5) that has a high (76%) sequenceidentity compared to the Bacillus subtilis ribosomal protein L2, andthus is referred to hereafter as rL2.

B. Production of Recombinant RAP (rL2)

[0147] To produce rL2, forward and reverse primers corresponding to the5′ and 3′ ends of rap gene with added 5′ NdeI and 3′ BamHI restrictionsites were designed based on the sequence of rap (underlined). Theseprimers, 5′ GAA TTC CAT ATG GCT ATT AAA AAG TAT AAG 3′ (nucleotides 1-21(SEQ IDNO:14)) and 5′ CGC GCG GAT CCT TAT TTT TTC TTA CGT CCA CG 3′(complement of nucleotides 840-819 (SEQ ID NO: 15)), were used amplifythe complete rap gene by PCR, using S. aureus chromosomal DNA as atemplate. Amplified DNA was digested by NdeI and BamHI and ligated intothe corresponding sites of pET14b vector (Novagen, Wis.) that possess asix histidine tag at the 5′ end of the inserted gene. Plasmid containingrap (pET2-5) was used to transform E. coli BL-21(DE3)pLysS (SBpET2-5).Induction of synthesis of recombinant protein was carried out byaddition of 1 mM IPTG to the culture and incubation for 3 hours. Cellswere harvested and washed once with 50 mM Tris buffer pH 7.9.

[0148] Recombinant His-rRAP protein was isolated using a nickel columnaccording to the manufacturer's instructions with some modifications(Xpress Systems Protein Purification, Invitrogen, Calif.). Cell pelletof 50 ml was resuspended in 10 ml binding buffer (20 mM sodium phosphatepH 7.8+0.5M NaCl) and sonicated (for 2 cycles of 15 sec pulses at themaximal level with 30 sec intervals) and then spun in a microcentrifuge.

[0149] The supernatant was loaded onto the pre-equilibrated nickelcolumn. Prior to loading, the column containing chelated Sepharose beadswas loaded with a charging buffer containing 50 mM NiCl₂, andequilibrated with binding buffer. The column was washed three times withfive volumes of binding buffer, followed by three washes with fivevolumes of 20 mM sodium phosphate+0.5M NaCl pH 7.8, then with bufferadjusted to pH 6. Recombinant protein was sequentially eluted from thecolumn using 5ml of the buffer containing 0.5, 1, 2, 3, and 4-Mimidazole.

[0150] Eluted fractions were extensively dialyzed overnight againstTRIS/EDTA buffer (5mM Tris pH 7.4, 0.5mM EDTA). Dialyzed fractions werelyophilized, and dried fractions were resuspended in 0.5 ml water, and30 μl applied on SDS 12.5% PAGE. Gel was western blotted, membranestained in ponceau to visualize proteins (FIG. 6A), blocked in 1% milk,and incubated with commercially available anti-his antibodies(Boehringer Mannheim) (FIG. 6B). As shown in FIGS. 6A and 6B, highestamounts of pure recombinant protein were eluted by 2M imidazole.

C. Vaccination of Animals with Recombinant RAP (rL2):

[0151] 4 week old female Balb/C mice (ten mice/group) were injectedsubcutaneously on days 0, 7, 21 with 50 μg rL2 (50 μg/50 μl PBS)together with 50 μl complete Freund's adjuvant on first injection andincomplete Freund's adjuvant on second and third injections. Controlanimals were injected with adjuvant/PBS only. Animals were challenged onday 35 with 2×10⁹ Smith Diffuse S. aureus (SD) prepared as describedbelow. Animals were observed daily for mortality, overall health anddevelopment of lesion. The size of the lesion was measured (area=0.5 7(length) (width).

[0152] Preparation of bacteria for challenge: Smith Diffuse S. aureuswas grown overnight at 37° C. on blood agar plates. Bacteria wassuspended in PBS at 2×10¹⁰ cells/ml. 2×10⁹ (100 μl) cells were injectedto vaccinated and control animals subcutaneously, together with 1 mgcytodex beads, to induce a local infection.

[0153] Antibody level as determined by ELISA. A drop of blood wascollected from the tip of the tail before the first vaccination and 10days after the third vaccination. ELISA plates were coated overnightwith 50 μl of 25 μg/ml antigen or with 3% BSA as a control. Wells werethen blocked with 3% BSA for 3 hrs at room temperature, and 50 μl sera(diluted 1:1000 in PBS) was applied for 2.5 hrs at room temperature.Unbound antibody was removed and wells were washed 5×2min with PBS with0.05% Tween 20. 50 μl peroxidase-conjugated anti-mouse antibody (Sigma)diluted 1:2000 with PBS/Tween was applied for 1 hr 37° C. Unboundantibody was removed, and wells were washed as above, and bound antibodywas detected by ABTS (Sigma) according to the manufacturer'sinstructions.

Results of Vaccination Experiments

[0154] Development of antibody to the antizen: All vaccinated animalsdeveloped an antibody titer (>1000) to the injected antigen. None of thecontrol animals had a detectable antibody level to the injected antigen.

[0155] Mortality post challenge: As shown in FIG. 7, of 10 controlanimals vaccinated with adjuvant only, 3 animals died within the firstday post challenge, and another mouse died on the second day. Of 10animals vaccinated with rL2, none died within the first day, one died onthe second day and another died on the third day.

[0156] Lesion: All surviving animals developed a lesion and its size wasdetermined on the fifth day post challenge. As shown in FIG. 8, theaverage lesion size of control animals was 7 cm², while the averagelesion size of animals vaccinated with rL2 was only 2.5 cm².

Conclusions

[0157] Animals vaccinated with rL2 had delayed mortality and a 50%reduction in mortality rate, and a 65% reduction in lesion size. Theseresults suggest that rL2 can confer protection to a S. aureus infection.Of note is the fact that the number of bacteria used for challenge wasexceptionally high and it is expected that if a lower number of bacteriawere present, protection level from infection could be higher.

[0158] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1 15 1 7 PRT Artificial Sequence MOD_RES (4) Cys, Trp or Ile, preferablyTrp 1 Tyr Lys Pro Xaa Thr Asn Phe 1 5 2 7 PRT Artificial SequenceMOD_RES (4) Cys, Trp or Ile, preferably Trp 2 Tyr Ser Pro Xaa Thr AsnPhe 1 5 3 10 PRT Artificial Sequence MOD_RES (7) Cys, Trp or Ile,preferably Trp 3 Ile Lys Lys Tyr Lys Pro Xaa Thr Asn Phe 1 5 10 4 10 PRTArtificial Sequence MOD_RES (7) Cys, Trp or Ile, preferably Trp 4 IleLys Lys Tyr Ser Pro Xaa Thr Asn Phe 1 5 10 5 21 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 5 tattcgccgtggaccaattt t 21 6 9 PRT Staphylococcus aureus 6 Ile Lys Lys Tyr Lys ProIle Thr Asn 1 5 7 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 7 Tyr Ser Pro Trp Thr Asn Phe 1 5 8 5 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide8 Pro Cys Thr Asn Phe 1 5 9 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 9 Tyr Lys Pro Ile Thr Asn Phe 1 510 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 10 Tyr Ser Pro Ile Thr Asn Phe 1 5 11 7 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 11 Tyr LysPro Trp Thr Asn Phe 1 5 12 840 DNA Staphylococcus sp. CDS (1)..(837) 12atg gct att aaa aag tat aag cca ata aca aat ggt cgt cgt aat atg 48 MetAla Ile Lys Lys Tyr Lys Pro Ile Thr Asn Gly Arg Arg Asn Met 1 5 10 15act tcg tta gat ttc gca gaa atc acg aaa act aca cct gaa aag tca 96 ThrSer Leu Asp Phe Ala Glu Ile Thr Lys Thr Thr Pro Glu Lys Ser 20 25 30 ttatta aaa ccg cta ccg aaa aaa gcg gga cgt aac aac caa ggt aaa 144 Leu LeuLys Pro Leu Pro Lys Lys Ala Gly Arg Asn Asn Gln Gly Lys 35 40 45 ttg actgta aga cac cat ggt ggt gga cac aaa cgt caa tac cgt gtt 192 Leu Thr ValArg His His Gly Gly Gly His Lys Arg Gln Tyr Arg Val 50 55 60 atc gat ttcaaa cgt aac aaa gat ggt atc aat gca aaa gtt gat tct 240 Ile Asp Phe LysArg Asn Lys Asp Gly Ile Asn Ala Lys Val Asp Ser 65 70 75 80 att caa tatgat cca aac cgc tca gca aac atc gct tta gtt gta tat 288 Ile Gln Tyr AspPro Asn Arg Ser Ala Asn Ile Ala Leu Val Val Tyr 85 90 95 gca gac ggt gaaaaa cga ata tat cat tgc att gct cct aaa gga tta 336 Ala Asp Gly Glu LysArg Ile Tyr His Cys Ile Ala Pro Lys Gly Leu 100 105 110 gaa gta ggt caaatc gtt gaa agt ggt gct gaa gct gac act aaa gtt 384 Glu Val Gly Gln IleVal Glu Ser Gly Ala Glu Ala Asp Thr Lys Val 115 120 125 ggt aac gca ttacca tta caa aac att cca gtt ggt aca gta gta cac 432 Gly Asn Ala Leu ProLeu Gln Asn Ile Pro Val Gly Thr Val Val His 130 135 140 aac atc gag cttaaa cct ggt aaa ggt gga caa atc gct cgt tca gct 480 Asn Ile Glu Leu LysPro Gly Lys Gly Gly Gln Ile Ala Arg Ser Ala 145 150 155 160 ggt gca agtgct caa gta ctt ggt aaa gaa ggt aaa tac gta tta atc 528 Gly Ala Ser AlaGln Val Leu Gly Lys Glu Gly Lys Tyr Val Leu Ile 165 170 175 aga tta agatct ggt gaa gtt cgt atg atc tta tct act tgc cgt gct 576 Arg Leu Arg SerGly Glu Val Arg Met Ile Leu Ser Thr Cys Arg Ala 180 185 190 aca atc ggtcaa gtt ggt aac cta caa cac gaa tta gtt aac gtt ggt 624 Thr Ile Gly GlnVal Gly Asn Leu Gln His Glu Leu Val Asn Val Gly 195 200 205 aaa gcc ggacgt tca aga tgg aaa ggt atc cgt cca aca gtt cgt ggt 672 Lys Ala Gly ArgSer Arg Trp Lys Gly Ile Arg Pro Thr Val Arg Gly 210 215 220 tct gta atgaac cct aac gat cac cca cac ggt ggt ggt gaa ggt cgt 720 Ser Val Met AsnPro Asn Asp His Pro His Gly Gly Gly Glu Gly Arg 225 230 235 240 gct cctatc ggt aga cca tct cca atg tca cca tgg ggt aaa cct acg 768 Ala Pro IleGly Arg Pro Ser Pro Met Ser Pro Trp Gly Lys Pro Thr 245 250 255 ctt ggtaag aaa act cgt cgt ggt aaa aaa tca tca gac aaa ctt atc 816 Leu Gly LysLys Thr Arg Arg Gly Lys Lys Ser Ser Asp Lys Leu Ile 260 265 270 gtt cgtgga cgt aag aaa aaa taa 840 Val Arg Gly Arg Lys Lys Lys 275 13 279 PRTStaphylococcus sp. 13 Met Ala Ile Lys Lys Tyr Lys Pro Ile Thr Asn GlyArg Arg Asn Met 1 5 10 15 Thr Ser Leu Asp Phe Ala Glu Ile Thr Lys ThrThr Pro Glu Lys Ser 20 25 30 Leu Leu Lys Pro Leu Pro Lys Lys Ala Gly ArgAsn Asn Gln Gly Lys 35 40 45 Leu Thr Val Arg His His Gly Gly Gly His LysArg Gln Tyr Arg Val 50 55 60 Ile Asp Phe Lys Arg Asn Lys Asp Gly Ile AsnAla Lys Val Asp Ser 65 70 75 80 Ile Gln Tyr Asp Pro Asn Arg Ser Ala AsnIle Ala Leu Val Val Tyr 85 90 95 Ala Asp Gly Glu Lys Arg Ile Tyr His CysIle Ala Pro Lys Gly Leu 100 105 110 Glu Val Gly Gln Ile Val Glu Ser GlyAla Glu Ala Asp Thr Lys Val 115 120 125 Gly Asn Ala Leu Pro Leu Gln AsnIle Pro Val Gly Thr Val Val His 130 135 140 Asn Ile Glu Leu Lys Pro GlyLys Gly Gly Gln Ile Ala Arg Ser Ala 145 150 155 160 Gly Ala Ser Ala GlnVal Leu Gly Lys Glu Gly Lys Tyr Val Leu Ile 165 170 175 Arg Leu Arg SerGly Glu Val Arg Met Ile Leu Ser Thr Cys Arg Ala 180 185 190 Thr Ile GlyGln Val Gly Asn Leu Gln His Glu Leu Val Asn Val Gly 195 200 205 Lys AlaGly Arg Ser Arg Trp Lys Gly Ile Arg Pro Thr Val Arg Gly 210 215 220 SerVal Met Asn Pro Asn Asp His Pro His Gly Gly Gly Glu Gly Arg 225 230 235240 Ala Pro Ile Gly Arg Pro Ser Pro Met Ser Pro Trp Gly Lys Pro Thr 245250 255 Leu Gly Lys Lys Thr Arg Arg Gly Lys Lys Ser Ser Asp Lys Leu Ile260 265 270 Val Arg Gly Arg Lys Lys Lys 275 14 30 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 14gaattccata tggctattaa aaagtataag 30 15 32 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 15 cgcgcggatccttatttttt cttacgtcca cg 32

We claim:
 1. An isolated RAP polypeptide having an amino acid sequenceof SEQ ID NO:13.
 2. An isolated nucleic acid molecule consisting of acoding sequence for the RAP polypeptide of claim
 1. 3. The isolatednucleic acid molecule of claim 2, wherein the coding sequence is SEQ IDNO:12 or a degenerate variant thereof.
 4. A method of treating aStaphylococcus infection, comprising: administering to a subject havinga Staphylococcus infection an amount of RAP effective to elicit anantibody response in the subject to treat the Staphylococcus infection.5. A vaccine comprising the RAP polypeptide of claim 1 or anantigenically effective portion thereof, and a pharmaceuticallyacceptable carrier.
 6. A method of preventing Staphylococcus aureusinfection, comprising administering to a subject a RAP polypeptide in anamount effective to elicit an immune response thereby creatingantibodies which bind RAP.